Gene Expression Analysis Of Bovine Oocytes And Preimpletation Embryons

Summary:Normal preimpletation development is characterized several cleavage division of the fertilized egg, activation of zygotic transcription, compaction and blastocoel formation, knowledge of genes and characterization of their expression patterns at different embryonic stages will provide critical information on genes which are important for normal preimplantation development, on the basis of the related research, by means of establishing bovine single embryo DDRT-PCR, dentials studies on gene expression in bovine embryo in vitro cultured had been carried out , bovine oocyte genes expression from ovaries with or without corpus luteum was studied at the same time. This is very important to study the elucidate embryo intrinsic development mechanism and anylize oocyte development competency.1. Establishment single bovine embryo DDRT-PCRThe method of single embryo RT-PCR plays an important role on investigating preimplantation development-related genes. selecting 4 cell embryo in vitro cultured, zone pellucida was lysised with acidic Tyrodes solution and embryos were lysised with lysis buffer, using single embryo and multiple embryos mRNA as templet, PCR amplification with radom primer and oligo-(dT) , the house keeping gene GAPDH PCR amplification with specific primer as control, amplification products was seprated on agrose gel electrophoresis(AGE) and polyacrylamide gel electrophoresis(PAGE). Multiple embryo result shows that ,the GAPDH was detected successfully in single embryo, Thus, method of single embryo RT-PCR is effective.That is multiple embryo and single ones has the same effects.2. Effect of corpus luteum on bovine oocyte genes expressionThe purpose of this study is to detect bovine oocyte genes from ovaries with or without corpus luteum Ovries had been collected from Lanzhou slaughterhouse which were divided into functional corpus luteum and no corpus luteum ,separating oocyte from ovries, mRNA of single oocyte lysates as templet with oligod(T) and radom primer,the oocyte genes had been amplified successfully by one-step reverse transcription polymerase chain reaction (RT-PCR), amplified products were detected with polyacrylamide gel electrophoresis(PAGE), there was no obvious difference in gel electrophoresis result, selecting the only one different gel electrophoresis belt to recover and purify ,PCR products was cloned into T-vector through sequencing and homologies analyzing ,it's Homologous with Early Embryo In Vitro Produced bemiv Bos taurus cDNA 3-mRNA sequence.3.Research on bovine preimpletation embryonic genesSelected 2-16 cell embryos in vitro cultured, using single embryo mRNA as templet, amplified products were detected with PAGE, there are 8 different belt in EGA stage ,different gel electrophoresis belt to recover and purify ,PCR products was cloned into T-vector through sequencing and homologies analyzing ,with Blast software 8 differential expression fragments were isolated from early bovine embryo, Homologous analysis indicated that,E1 sequence is Homologous with M.musculus mRNA for Oct-4 protein ;E2 sequence is Homologous with Bos taurus similar to Cyclin-dependent kinases regulatory subunit 1;E3 sequence is Homologous with Bos taurus THAP domain containing 9; E4 sequence is Homologous with mg98c01.r1 Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA ;E5 sequence is Homologous with Bos taurus high-mobility group box 2; E6 sequence is Homologous with Mus musculus 10 days embryo whole body cDNA ; E7 sequence is Homologous with Bos taurus similar to Chromosome 11 open reading frame; E8 sequence is Homologous with Bos taurus Sec61 gamma subunit (SEC61G) these genes related to cell split ,cleavage, cell recycle regulation, then these genes play important role in early embryo development.